The present invention relates in general to systems for the microbial production of biotin and in particular to systems wherein at least a part of the biotin operon is present on a plasmid within a biotin retention-deficient mutant host cell.
Biotin, also known as vitamin H, is probably an essential component of all cells. Some microorganisms, including baker's yeast, and all animals (except the protozoan Tetrahymena) are unable to synthesize biotin effectively and must therefore obtain biotin from their environment in order to survive.
Despite its usefulness in promoting the growth of baker's yeast and as a human and animal food additive, biotin is very expensive to manufacture by presently available, chemical synthetic methods. Furthermore, although beet molasses (containing 0.015-0.15 .mu.grams of biotin per gram) or other natural sources of biotin may be used to supplement synthetic biotin, there exists a need for other sources.
Due to the ready availability of information regarding the genetic constitution of certain microorganisms which have been reported to contain relatively high concentrations of biotin, a capability for performing genetic manipulations on those microorganisms has developed. It has been reported, for example, that certain chromosomal genes which encode enzymes of the pathway for biotin synthesis may be isolated, amplified and reinserted into host cells of the bacterium Escherichia coli (E. coli).
More specifically, Mukherjee, et al. in "Plasmids and Transposons," Stuttard, et. al., eds., Academic Press, New York (1980), 379-386 reported isolation of the biotin operon of the E. coli K-12 strain from a transducing bacteriophage by means of EcoRI enzyme digestion. A restriction fragment was inserted into a DNA plasmid (pMB8) which was used to transform E. coli host cells to provide multiple "extra" copies of the biotin operon genes in these hosts. Mukherjee, et al., however fails to teach or even suggest the use of a biotin retention-deficient mutant genotype host cell. Although enhancement of excretion over a biotin prototroph ("wild type") was reported, this recombinant system has not been applied to large scale commercial production of biotin by fermentation of transformed host cells.